Promoter analysis of seed-specific genes has a rich history (reviewed in Goldberg et al. (1989) Cell, 56; 149-160; Thomas (1993) Plant Cell, 5; 1401-1410). This stems from the observation that no plant gene is more tightly regulated in terms of spatial expression than those encoding seed storage proteins. Many seed storage protein genes have been cloned from diverse plant species, and their promoters have been analyzed in detail (Thomas, 1993). In these experiments promoter elements, which constitute the 5'-upstream regulatory regions, were functionally defined by their ability to confer seed-specific expression of the bacterial .beta.-glucuronidase (GUS) reporter gene in transgenic plants (Bogue et al. (1990) Mol. Gen. Genet., 222; 49-57; Bustos et al. (1989) Plant Cell, 1; 839-853). Results of this work initiated efforts to functionally define cis-elements to these genes that are critical for conferring seed-specific expression.
Later experiments involved construction of deletion mutants consisting of target promoters fused to the GUS-reporter gene. Analysis of these constructs in transgenic plants allowed researchers to define regions within each promoter that are critical to its overall regulation (Bustos et al. (1991) EMBO J., 10; 1469-1479; Chung (1995) Ph.D. Dissertation, Texas A&M University; Nunberg et al. (1994) Plant Cell, 6; 473-486). A general conclusion from this work is that the promoter proximal region contributes primarily to the gene's tissue specificity with more distal regions being responsible for modulating expression levels (Thomas, 1993). In addition to this, several groups have identified and characterized specific cis-regulatory elements, in both the promoter proximal region (PPR) and more distal regions, which interact with DNA binding proteins (Bustos et al., 1989; Chung, 1995; Jordano et al. (1989) Plant Cell, 1; 855-866; Nunberg et al., 1994). The functional significance of these regulatory elements varies from gene to gene.
In some cases, cis-regulatory elements have been mapped and the trans-acting factors which confer functionality have been cloned. For example, elements that allow the wheat EM-gene to respond to the plant hormone abscisic acid (ABA) have been defined. This work led to the identification of a DNA binding protein which mediates this response (Guiltinan et al. (1990) Science, 250; 267-271; Marcotte et al. (1989) Plant Cell, 1; 969-976). Putative ABA responsive elements have also been mapped in the sunflower helianthinin promoter HaG3-D and the carrot Dc3 promoter (Chung, 1995; Nunberg et al., 1994). Alone these elements act as positive elements in response to ABA. Regulation is restricted to the embryo, however, in the presence of each gene's promoter proximal region (Thomas, 1993).
Despite considerable effort, the cis-regulatory elements which contribute to a promoter's seed-specificity remain elusive (Chung, 1995; Li (1995) Ph.D. Dissertation, Texas A&M University). Recent work on the carrot Dc3 promoter proximal region has identified two bZIP genes that functionally interact with critical cis-elements (Kim et al. (1997) Plant J., 11; 1237-1251). This.work has increased the understanding of seed-specific gene expression but it has also revealed that seed-specific gene regulation is complex.
In Arabidopsis thaliana, the promoters driving the expression of four members of the 2S albumin gene family have been analyzed in detail. The data indicate that each promoter is capable of conferring seed specific expression of a reporter gene in transgenic plants. Each promoter, however, confers slightly different spatial accumulation of the reporter in the developing seed. Thus, each family member contributes to the overall accumulation of the 2S albumins in the developing embryo. This is not unusual behavior for small gene families in plants (Lam et al. (1995) Plant Cell, 7; 887-898; Conceicao et al. (1994) Plant J., 5; 493-505; Sjodahl et al. (1993) Plant Mol. Biol., 23; 1165-1176; Pang et al. (1988) Plant Mol. Biol., 11; 805-820). In such cases, each member is capable of functionally complementing the others. The expression of each member is under different regulatory control leading to unique expression patterns. This appears to be a widespread gene regulatory mechanism in plants.
Little information is available on the contribution of a gene's untranslated elements to overall gene activity. In particular, the role of a gene's 5'-transcribed but untranslated region has never been fully investigated and is therefore not well understood. It is clear from the analysis of several plant genes, that these regions can significantly contribute to overall gene activity (Fu et al. (1995b) Plant Cell, 7; 1395-1403; Larkin et al. (1993) Plant Cell, 5; 1739-1748; Sieburth et al. (1997) Plant Cell, 9; 355-365). The general role of these regions, if any, is not known. This is mainly due to the observation that a gene's promoter, defined as the gene's 5'-untranscribed region which consists of 1.0-1.5 kb of 5'-upstream sequence, is necessary and sufficient to confer spatial and temporal expression of the GUS reporter gene in transgenic plants. It may or may not be sufficient to account for overall gene activity. A general comparison of these regions reveals little or no conservation between diverse genes, and a similar observation has been made with respect to promoter elements as well (Conceicao et al., 1994).
Despite the uncertainties associated with seed-specific regulatory elements, there is substantial interest in identification and isolation of such regulatory elements for use in manipulating expression of both native and heterologous genes in plant seeds. For example, well-defined seed specific regulatory elements are useful in manipulating fatty acid synthesis or lipid metabolism genes in plant seeds. Other important agronomic traits such as herbicide and pesticide resistance, and drought tolerance may also be altered in the plant seed by transforming plants with appropriate heterologous genes under the control of well-defined seed-specific promoters and cis regulatory elements.
The present invention provides regulatory elements including promoters and 5' untranslated regions from two seed-specific genes designated AtS1 and AtS3. The regulatory elements may be used with any native or heterologous gene or portion thereof for expression of a corresponding gene product in a plant seed.